In order to understand the biological and functional relationship between mammographic density and breast cancer risk, experimental models are needed to investigate key features in a controlled context. We propose that the mechanistic link between dense breasts and breast cancer risk is that the biological processes that lead to increased breast density can promote malignant progression by initiated cells in the adjacent epithelium. We hypothesize that features of increased stroma, remodeled extracellular matrix (ECM) and molecular markers in mammographically dense breasts are indicative of an 'activated'stroma. Part of the normal continuum of stromal phenotypes, activated stroma has similarities to the stromas formed during morphogenesis and wound healing. Our experimental studies and those of others have demonstrated that atypical stroma can become an active participant in cancer progression. Project 4 will develop two activated stroma (AS) mouse models to test these hypotheses. An exogenous agent, ionizing radiation (IR), and a genetically engineered mouse expressing constitutively active transforming growth factor beta1 (TGF-beta) on a fibroblast-specific promoter (FSP-Tgf-beta (223-225)) will be used to generate mammary AS. Radiotherapy for breast cancer transiently increases mammographic density. We have shown that IR induces mammary stromal ECM remodeling in mice similar in character to that found in dense breast tissue. Together these observations provide the rationale that IR may be used as an experimental tool to induce a mouse mammary stroma similar to that in dense breasts. We have shown that TGF-beta1 mediates radiation-induced ECM remodeling and is involved in ovarian hormone regulation of normal breast development. Others have demonstrated that TGF-beta is deregulated during cancer progression. Certain Tgf-beta1 gene polymorphisms are associated with increased breast cancer risk. Thus, TGF-beta fulfills the criteria of being a genetic trait that predisposes certain women to breast cancer as well as a hormonally regulated growth factor whose actions affect mammary tissue composition. The mammary AS produced by IR and the FSP-Tgf-beta(223-225) transgenic mouse models will be compared to the features of dense breast tissue determined in Project 2. We will use these models to answer the following: 1. How do the known breast density modulators of parity, age and hormonal status affect the specific features of AS;2. Is epithelial proliferation, apoptosis, or morphogenesis affected by stromal activation;and 3. Does AS promote neoplastic progression? We will evaluate markers that are identified in Project 2 and tested in Project 3 in the mouse models to define commonalities that can be used as benchmarks of critical processes. Mouse models permit evaluation of how important lifestyle variables contribute to the expression of a given marker.